We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 mu m, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-mu m segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, similar to 2 mu m) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20 degrees, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general. (c) 2012 Elsevier Inc. All rights reserved.